Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat Victoria Brusa, Luc´ıa Galli, Luciano H. Linares, Emanuel E. Ortega, Juan P. Lir´on, Gerardo A. Leotta PII: DOI: Reference:

S0167-7012(15)30069-5 doi: 10.1016/j.mimet.2015.09.013 MIMET 4743

To appear in:

Journal of Microbiological Methods

Received date: Revised date: Accepted date:

14 June 2015 22 September 2015 23 September 2015

Please cite this article as: Brusa, Victoria, Galli, Luc´ıa, Linares, Luciano H., Ortega, Emanuel E., Lir´on, Juan P., Leotta, Gerardo A., Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat, Journal of Microbiological Methods (2015), doi: 10.1016/j.mimet.2015.09.013

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ACCEPTED MANUSCRIPT REVISED Development and Validation of Two SYBR Green PCR Assays and a Multiplex Real-

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Time PCR for the Detection of Shiga Toxin-Producing Escherichia coli in Meat

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Victoria Brusa1, 2, Lucía Galli2, Luciano H. Linares1, Emanuel E. Ortega1, Juan P. Lirón2,

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Gerardo A. Leotta2*

Laboratorio de Microbiología de Alimentos, Facultad de Ciencias Veterinarias UNLP, La

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Plata, Argentina.

IGEVET - Instituto de Genética Veterinaria “Ing. Fernando N. Dulout” (UNLP-

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CONICET LA PLATA), Facultad de Ciencias Veterinarias UNLP, La Plata, Argentina.

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Short title: SYBR Green and RT-PCR for STEC detection in meat

*Corresponding author. E-mail address: [email protected] (G.A. Leotta). IGEVET - Instituto de Genética Veterinaria “Ing. Fernando N. Dulout” (UNLP-CONICET LA PLATA), Facultad de Ciencias Veterinarias UNLP. Av. 60 y 118, CC: 296. La Plata (1900), Provincia de Buenos Aires, Argentina

ACCEPTED MANUSCRIPT ABSTRACT Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne

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pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-

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time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one

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hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification

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control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-

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STEC strains were used. Naturally contaminated ground beef samples (n=103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stx-positive

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samples were processed for STEC isolation. In the intra-laboratory validation, each PCR

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obtained a 1×102 CFU mL-1 limit of detection and 100% inclusivity and exclusivity. The

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same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa=0.758 and 0.801 (P